Experiments will be carried out to analyze the role of DNA methylation in the regulation of gene expression in mammalian cells in culture. The experiments will utilize the techniques of biochemistry, somatic cell genetics, and recombinant DNA to determine the effects of altering methylation patterns on gene expression. The studies will be carried out in 2 different systems: (1) transformed mouse L cells containing a fragment of Herpes Simplex virus DNA that includes the viral gene for thymidine kinase (TK), and (2) Chinese hamster V79 cells which contain an inactive phenylalanine hydroxylase (PH) gene. We have already obtained evidence indicating that the expression of both the viral TK gene and the hamster PH gene can be induced by 5-azacytidine, a compound which causes DNA hypomethylation by substituting for cytosine in DNA. We have also shown an inverse correlation between the expression of the viral TK gene and its state of methylation in a series of transformed lines. Through the analysis of the biological and molecular effects of treating cells with inducers of hypothylation, we will attempt to determine in what ways and by which mechanisms does DNA methylation effect gene expression. Using available molecular probes for the viral TK gene, these studies will include the determination of specific methylation patterns in both structural and regulatory sequences and the analysis of the relationship between specific methylation patterns and gene expression. We will also attempt to clone the hamster PH gene, so that detailed molecular experiments on DNA methylation can be performed in that system as well. The two biological systems to be studied pose different types of questions, as one system (TK) involves the expression of an exogenous viral gene incorporated into the cellular genome, while the other system (PH) involves a normal cellular gene whose expression is rigidly controlled in differentiation. The results of these studies should provide information on the mechanisms by which cellular and exogenous genes are regulated in mammalian cells, both in normal development and in disease states. In addition, the studies of the PH system should progress towards a detailed molecular analyses of the human disease phenylketonuria.